Inbred Pepper Line PPL1501

ABSTRACT

One embodiment relates to seed and plants of inbred pepper line PPL1501. Another embodiment relates to the plants, seeds and tissue cultures of inbred pepper line PPL1501, and to methods for producing a pepper plant produced by crossing such plants with themselves, with another pepper plant, such as a plant of another genotype, or with vegetatively propagating said plant. Another embodiment further relates to seeds and plants produced by such crossing. Further embodiments relate to parts of such plants, including the fruit and gametes of such plants.

BACKGROUND

The embodiments recited herein relates to a novel and distinct inbredpepper (Capsicum annuum) line designated PPL1501, and to the seeds,plant parts, and tissue culture produced by that inbred pepper line. Theembodiments further relate to food products produced from inbred pepperline PPL1501, such as, but not limited to, fruit, powders, sauces, andsalsas. All publications cited in this application are hereinincorporated by reference.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY

It is to be understood that the embodiments include a variety ofdifferent versions or embodiments, and this Summary is not meant to belimiting or all-inclusive. This Summary provides some generaldescriptions of some of the embodiments, but may also include some morespecific descriptions of other embodiments.

An embodiment provides an inbred pepper line designated PPL1501. Anotherembodiment relates to the seeds of inbred pepper line PPL1501, to theplants of inbred pepper line PPL1501 and to methods for producing apepper plant produced by crossing inbred pepper line PPL1501 with itselfor another inbred pepper line, and the creation of variants bymutagenesis or transformation of inbred pepper line PPL1501.

Any such methods using inbred pepper line PPL1501 are a furtherembodiment: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using inbred pepper linePPL1501 as at least one parent are within the scope of the embodiments.Advantageously, inbred pepper line PPL1501 could be used in crosses withother, different pepper plants to produce first generation (F₁) pepperhybrid seeds and plants with superior characteristics.

Another embodiment provides for single or multiple gene converted plantsof inbred pepper line PPL1501. The transferred gene(s) may be a dominantor recessive allele. The transferred gene(s) may confer such traits asherbicide tolerance, insect tolerance, tolerance for bacterial, fungal,or viral disease, male fertility, male sterility, enhanced nutritionalquality, environmental stress tolerance, modified carbohydratemetabolism, modified yield, modified glycoalkaloid content, andindustrial usage. The gene may be a naturally occurring pepper gene or atransgene introduced through genetic engineering techniques.

Another embodiment provides for regenerable cells for use in tissueculture of inbred pepper line PPL1501. The tissue culture may be capableof regenerating plants having all the physiological and morphologicalcharacteristics of the foregoing pepper plant, and of regeneratingplants having substantially the same genotype as the foregoing pepperplant. The regenerable cells in such tissue cultures may be embryos,protoplasts, meristematic cells, callus, pollen, leaves, ovules,anthers, cotyledons, hypocotyl, pistils, roots, root tips, flowers,seeds, plant, petiole, or stems. Still a further embodiment provides forpepper plants regenerated from the tissue cultures of inbred pepper linePPL1501.

Another embodiment relates to a method of vegetatively propagatinginbred pepper line PPL1501 comprising the steps of: (a) collectingtissue capable of being propagated from the plant; (b) cultivating saidtissue to obtain proliferated shoots; and (c) rooting said proliferatedshoots to obtain rooted plantlets.

Another embodiment provides for a method for producing a seed of apepper plant derived from inbred pepper line PPL1501 comprising thesteps of: (a) crossing the pepper plant with itself or a second pepperplant, and (b) allowing seed of an inbred PPL1501-derived pepper plantto form.

Further embodiments provide for a method of producing food or feedcomprising: (a) obtaining a plant of inbred pepper line PPL1501, whereinthe plant has been cultivated to maturity, and (b) collecting at leastone pepper from the plant.

Another embodiment provides for the genetic complement of inbred pepperline PPL1501. The phrase “genetic complement” is used to refer to theaggregate of nucleotide sequences, the expression of which sequencesdefines the phenotype of, in the present case, a pepper plant, or a cellor tissue of that plant. A genetic complement thus represents thegenetic makeup of a cell, tissue or plant, and a hybrid geneticcomplement represents the genetic make-up of a hybrid cell, tissue orplant. Thus another embodiment provides for pepper plant cells that havea genetic complement in accordance with the pepper plant cells disclosedherein, and seeds and plants containing such cells.

As used herein, “at least one,” “one or more,” and “and/or” areopen-ended expressions that are both conjunctive and disjunctive inoperation. For example, each of the expressions “at least one of A, Band C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “oneor more of A, B, or C” and “A, B, and/or C” means A alone, B alone, Calone, A and B together, A and C together, B and C together, or A, B andC together.

Various embodiments are set forth in the Detailed Description asprovided herein and as embodied by the claims. It should be understood,however, that this Summary does not contain all of the aspects andembodiments, is not meant to be limiting or restrictive in any manner,and that embodiment(s) as disclosed herein is/are understood by those ofordinary skill in the art to encompass obvious improvements andmodifications thereto.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Embryo. The embryo is the small plant contained within a mature seed.

Gene. Gene refers to a segment of nucleic acid. A gene can be introducedinto a genome of a species, whether from a different species or from thesame species, using transformation or various breeding methods.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Locus. Locus or loci (plural) refers to a position in the genome for agene, SNP, mutation, etc.

Plant Parts. Plant parts (or a pepper plant, or a part thereof) includesbut is not limited to, regenerable cells in such tissue cultures may beembryos, protoplasts, meristematic cells, callus, pollen, leaves,ovules, anthers, cotyledons, hypocotyl, pistils, roots, root tips,flowers, fruit, seeds, plant, petiole, or stems.

Progeny. Progeny includes an Fi pepper plant produced from the cross oftwo pepper plants where at least one plant includes inbred pepper linePPL1501 and progeny further includes, but is not limited to, subsequentF₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, and F₁₀ generational crosses with therecurrent parental line.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Refers to the development of a plant from tissue culture.

RHS. RHS refers to the Royal Horticultural Society color reference.

Single Gene Converted (Conversion). Single gene converted (conversion)plants refers to plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique or via genetic engineering.

Wall Thickness. Wall thickness refers to the diameter of the fruit wall.Wall thickness is measured by cutting the fruit in half longitudinallyand measuring the thickness (mm) on the thinnest part of each side ofthe wall of each fruit. Wall thickness is important to both bell peppergrowers and shippers because fruit having thick walls are more likely toship better without the fruit wall cracking.

DETAILED DESCRIPTION

Inbred pepper line PPL1501 originated from a private breeding program inFt. Myers, Florida. The open pollinated F₂ seed was derived from ahybrid pepper variety obtained in 2012 by the breeder, and then advancedto F₄ generation via single plant selection based on phytopathology livetest results as well as horticultural traits including, but not limitedto, plant type, fruit size, fruit shape and fruit color. Inbred pepperline PPL1501 produces medium anthocyanin-less plant bearing large toextra-large size blocky-half long sweet bell pepper fruits. The firmfruits can be 3 to 4 lobed maturing from medium-dark green to red color.The line is resistant to Tobacco Mosaic Virus race (Tm0), andXanthomonas compestris pv. vesicatoria (Xcv) races 0 to 10.

Inbred pepper line PPL1501 has shown uniformity and stability, asdescribed in the following variety description information. Inbredpepper line PPL1501 was tested for uniformity and stability a sufficientnumber of generations with careful attention to uniformity of plant typeand has been increased with continued observation for uniformity.

Inbred pepper line PPL1501 has the following morphologic and othercharacteristics based primarily on data collected in Ft. Myers, Florida.

TABLE 1A TABLE 1: VARIETY DESCRIPTION INFORMATION (COMPRISED OF TABLES1A AND 1B) Characteristic PPL1501 General Fruit Type Bell AdaptationMost U.S. Areas Maturity -Transplant to Green Stage 75 days Maturity-Transplant to Red or Yellow Stage 110 days Maturity - Seedling to GreenStage 120 days Maturity - Seedling to Red or Yellow Stage 155 days PlantHabit Compact Plant Attitude Semi-Erect Plant Height (from soil to topof plant) 58.0 cm Plant Width 52.0 cm Length of Third Internode (fromsoil surface) 23.0 cm Basal Branches Few (2 to 3) Branch FlexibilityWillowy/Rigid Stem Strength Intermediate Mature Leaf Shape LanceolateLeaf Length 125.0 mm Leaf width 60.0 mm Petiole Length 40.0 mm Leaf andStem Pubescence Absent Margin Undulation Very Weak Blistering Weak LeafColor Dark Green Number of Flowers per Leaf Axil 1 Number of Calyx Lobes0 Number of Petals 7 Flower Diameter 150.0 mm Corolla Color WhiteCorolla Throat Markings Yellow (Tan) Anther Color Yellow Style LengthExceeds Stamen Self-Incompatibility Absent Immature Fruit Color LightGreen Mature Fruit Color Red Pungency Sweet Fruit Glossiness ModerateSurface Smoothness Smooth Fruit Position Upright/Horizontal Calyx ShapeSaucer-Shaped Calyx Diameter 100.0 mm Fruit Length 120.0 mm FruitDiameter at Calyx Attachment 95.0 mm Fruit Diameter at Mid-Point 100.0mm Wall Thickness at Mid-Point 6.0 mm Average Number of Fruit/Plant 6Fruit Base Shape Cupped Fruit Apex Shape Blunt Fruit Shape Bell FruitShape (Longitudinal Section) Rectangular Fruit Shape (Cross Section)Quadrangular Fruit Set Concentrated Interloculary Grooves Deep AverageNumber of Locules 4 Pedicel Length 45.0 mm Pedicel Thickness 6.0 mmPedicel Shape Curved Pedicel Cavity Present Depth of Pedicel Cavity 20.0mm Seed Color Yellow Anthocyanin Presence in Seedling Hypocotyl AbsentAnthocyanin Presence in Stem Absent Anthocyanin Presence in Node AbsentAnthocyanin Presence in Leaf Absent Anthocyanin Presence in PedicelAbsent Anthocyanin Presence in Calyx Absent Anthocyanin Presence inFruit Absent Vigor 3

TABLE 1B Disease PPL1501 Xanthomonas compestris pv. vesicatoria (Xcv)races 0 to 10 Resistant Tobacco Mosaic Virus ResistantBreeding With Inbred pepper line PPL1501

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable variety. This approach hasbeen used extensively for breeding disease-resistant varieties. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial varieties; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardvariety. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of pepper breeding is to develop new and superior peppervarieties and hybrids. The breeder initially selects and crosses two ormore parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selection, selfing and mutations.

Using Inbred pepper line PPL1501 to Develop other Pepper Varieties

Pepper varieties such as inbred pepper line PPL1501 are typicallydeveloped for fresh consumption. However, pepper varieties such asinbred pepper line PPL1501 also provide a source of breeding materialthat may be used to develop new pepper varieties. Plant breedingtechniques known in the art and used in a pepper breeding programinclude, but are not limited to, recurrent selection, bulk selection,mass selection, backcrossing, pedigree breeding, open pollinationbreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, making double haploids,transformation, and gene editing. These techniques can be usedsingularly or in combinations. The development of pepper varieties in abreeding program requires, in general, the development and evaluation ofhomozygous varieties. There are many analytical methods available toevaluate a new variety. The oldest and most traditional method ofanalysis is the observation of phenotypic traits, but genotypic analysismay also be used.

Additional Breeding Methods

One embodiment is directed to methods for producing a pepper plant bycrossing a first parent pepper plant with a second parent pepper plant,wherein the first or second pepper plant is the pepper plant from inbredpepper line PPL1501. Further, both first and second parent pepper plantsmay be from inbred pepper line PPL1501. Any plants produced using inbredpepper line PPL1501 as at least one parent are also within the scope ofthe embodiments. These methods are well known in the art and some of themore commonly used breeding methods are described herein. Descriptionsof breeding methods can be found in one of several reference books(e.g., Allard, Principles of Plant Breeding (1960); Simmonds, Principlesof Crop Improvement (1979); Sneep, et al. (1979); Cooper, S. G., D.S.Douches and E. J. Grafius. 2004.

The following describes breeding methods that may be used with inbredpepper line PPL1501 in the development of further pepper plants. Onesuch embodiment is a method for developing an inbred pepper line PPL1501progeny plant in a pepper breeding program comprising: obtaining thepepper plant, or a part thereof, of inbred pepper line PPL1501,utilizing said plant, or plant part, as a source of breeding material,and selecting an inbred pepper line PPL1501 progeny plant with molecularmarkers in common with inbred pepper line PPL1501 and/or withmorphological and/or physiological characteristics selected from thecharacteristics listed in Table 1. Breeding steps that may be used inthe pepper plant breeding program include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example, SSR markers), and the making ofdouble haploids may be utilized.

Another method involves producing a population of inbred pepper linePPL1501 progeny pepper plants, comprising crossing inbred pepper linePPL1501 with another pepper plant, thereby producing a population ofpepper plants which derive 50% of their alleles from inbred pepper linePPL1501. A plant of this population may be selected and repeatedlyselfed or sibbed with an inbred pepper line resulting from thesesuccessive filial generations. One embodiment is the inbred pepper lineproduced by this method and that has obtained at least 50% of itsalleles from inbred pepper line PPL1501.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see, Fehr and Walt, Principles of VarietyDevelopment, pp. 261-286 (1987). Thus, embodiments include inbred pepperline PPL1501 progeny pepper plants comprising a combination of at leasttwo inbred pepper line PPL1501 traits selected from the group consistingof those listed in Table 1 and a combination of traits listed in theSummary, so that said progeny pepper plant is not significantlydifferent for said traits than inbred pepper line PPL1501 as determinedat the 5% significance level when grown in the same environmentalconditions. Using techniques described herein, molecular markers may beused to identify said progeny plant as an inbred pepper line PPL1501progeny plant. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions. Once such avariety is developed, its value is substantial since it is important toadvance the germplasm base as a whole in order to maintain or improvetraits such as yield, disease tolerance, pest tolerance, and plantperformance in extreme environmental conditions.

Progeny of inbred pepper line PPL1501 may also be characterized throughtheir filial relationship with inbred pepper line PPL1501, as forexample, being within a certain number of breeding crosses of inbredpepper line PPL1501. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a self or a sibcross, which is made to select among existing genetic alleles. The lowerthe number of breeding crosses in the pedigree, the closer therelationship between inbred pepper line PPL1501 and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4, or 5 breeding crosses of inbred pepper line PPL1501.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asinbred pepper line PPL1501 and another inbred pepper line having one ormore desirable characteristics that is lacking or which complementsinbred pepper line PPL1501. If the two original parents do not provideall the desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations, the heterozygous condition gives way to homogeneousvarieties as a result of self-pollination and selection. Typically inthe pedigree method of breeding, five or more successive filialgenerations of selfing and selection is practiced: F₁ to F₂; F₂ to F₃;F₃ to F₄; F₄ to F₅; etc. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped variety. Preferably, the developed variety compriseshomozygous alleles at about 95% or more of its loci.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous variety orinbred line which is the recurrent parent. The source of the trait to betransferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., variety) and the desirable trait transferred from thedonor parent. This is also known as single gene conversion and/orbackcross conversion.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. It may be necessary to introduce a test of the progeny todetermine if the desired characteristic has been successfullytransferred.

A backcross conversion of inbred pepper line PPL1501 occurs when DNAsequences are introduced through backcrossing, with inbred pepper linePPL1501 utilized as the recurrent parent. Both naturally occurring andtransgenic DNA sequences may be introduced through backcrossingtechniques. A backcross conversion may produce a plant with a trait orlocus conversion in at least two or more backcrosses, including at least2 crosses, at least 3 crosses, at least 4 crosses, at least 5 crosses,and the like. Molecular marker assisted breeding or selection may beutilized to reduce the number of backcrosses necessary to achieve thebackcross conversion. For example, see, Frisch M. et al,“Marker-Assisted Backcrossing for Simultaneous Introgression of TwoGenes” Crop Science Society of America, pp 1716-1725 (2001) andOpenshaw, S. J., et al., “Marker-assisted Selection in BackcrossBreeding, Proceedings Symposium of the Analysis of Molecular Data” CropScience Society of America, Corvallis, Oreg. (August 1994), where it wasdemonstrated that a backcross conversion could be made in as few as twobackcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. Desired traits that may be transferred through backcrossconversion include, but are not limited to, sterility (nuclear andcytoplasmic), fertility restoration, nutritional enhancements, droughttolerance, nitrogen utilization, low phytate, industrial enhancements,disease tolerance (bacterial, fungal, or viral), insect tolerance, andherbicide tolerance. In addition, an introgression site itself, such asan FRT site, Lox site, or other site specific integration site, may beinserted by backcrossing and utilized for direct insertion of one ormore genes of interest into a specific plant variety. In someembodiments, the number of loci that may be backcrossed into inbredpepper line PPL1501 is at least 1, 2, 3, 4, or 5, and/or no more than 6,5, 4, 3, or 2. A single locus may contain several transgenes, such as atransgene for disease tolerance that, in the same expression vector,also contains a transgene for herbicide tolerance. The gene forherbicide tolerance may be used as a selectable marker and/or as aphenotypic trait. A single locus conversion of site specific integrationsystem allows for the integration of multiple genes at the convertedloci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing eachbackcross generation to determine which plants carry the recessivealleles unless molecular markers are available to track the gene.Recessive traits may require additional progeny testing in successivebackcross generations to confirm the presence of the locus of interest.The last backcross generation is usually selfed to give pure breedingprogeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, “Breeding Field Crops”p. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant, and easily recognized traits.

One process for adding or modifying a trait or locus in inbred pepperline PPL1501 comprises crossing inbred pepper line PPL1501 plants grownfrom inbred pepper line PPL1501 seed with plants of another inbredpepper line that comprise the desired trait or locus, selecting Fiprogeny plants that comprise the desired trait or locus to produceselected Fi progeny plants, crossing the selected progeny plants withthe inbred pepper line PPL1501 plants to produce backcross progenyplants, selecting for backcross progeny plants that have the desiredtrait or locus and the morphological characteristics of inbred pepperline PPL1501 to produce selected backcross progeny plants, andbackcrossing to inbred pepper line PPL1501 three or more times insuccession to produce selected fourth or higher backcross progeny plantsthat comprise said trait or locus. The modified inbred pepper linePPL1501 may be further characterized as having the physiological andmorphological characteristics of inbred pepper line PPL1501 listed inTable 1 as determined at the 5% significance level when grown in thesame environmental conditions and/or may be characterized by percentsimilarity or identity to inbred pepper line PPL1501 as determined bySSR markers. The above method may be utilized with fewer backcrosses inappropriate situations, such as when the donor parent is highly relatedor markers are used in the selection step. Desired traits that may beused include those nucleic acids known in the art, some of which arelisted herein, that will affect traits through nucleic acid expressionor inhibition. Desired loci include the introgression of FRT, Lox, andother sites for site specific integration, which may also affect adesired trait if a functional nucleic acid is inserted at theintegration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny pepper seed byadding a step at the end of the process that comprises crossing inbredpepper line PPL1501 with the introgressed trait or locus with adifferent pepper plant and harvesting the resultant first generationprogeny pepper seed.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques well-known in the art. Single genetraits may or may not be transgenic. Examples of these traits include,but are not limited to, herbicide tolerance, insect tolerance, tolerancefor bacterial, fungal, or viral disease, male fertility, male sterility,enhanced nutritional quality, modified carbohydrate metabolism, modifiedyield, modified glycoalkaloid content, and industrial usage

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodagronomic characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, aninbred pepper line may be crossed with another variety to produce afirst generation progeny plant. The first generation progeny plant maythen be backcrossed to one of its parent varieties to create a BC₁ orBC₂. Progeny are selfed and selected so that the newly developed varietyhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the nonrecurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newpepper varieties.

Therefore, an embodiment of the present disclosure is a method of makinga backcross conversion inbred pepper line PPL1501, comprising the stepsof crossing a plant of inbred pepper line PPL1501 with a donor plantcomprising a desired trait, selecting an F₁ progeny plant comprising thedesired trait, and backcrossing the selected F₁ progeny plant to a plantof inbred pepper line PPL1501 to produce BC₁, BC₂, BC₃, etc. This methodmay further comprise the step of obtaining a molecular marker profile ofinbred pepper line PPL1501 and using the molecular marker profile toselect for a progeny plant with the desired trait and the molecularmarker profile of inbred pepper line PPL1501. In one embodiment, thedesired trait is a mutant gene, gene, or transgene present in the donorparent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Inbred pepper line PPL1501 is suitablefor use in a recurrent selection program. The method entails individualplants cross pollinating with each other to form progeny. The progenyare grown and the superior progeny selected by any number of selectionmethods, which include individual plant, half-sib progeny, full-sibprogeny, and selfed progeny. The selected progeny are cross pollinatedwith each other to form progeny for another population. This populationis planted and again superior plants are selected to cross pollinatewith each other. Recurrent selection is a cyclical process and thereforecan be repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtainnew varieties for commercial or breeding use, including the productionof a synthetic variety. A synthetic variety is the resultant progenyformed by the intercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Mutation Breeding

Mutation breeding is another method of introducing new traits intoinbred pepper line PPL1501. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil)), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in Fehr,“Principles of Variety Development,” Macmillan Publishing Company(1993). In addition, mutations created in other pepper plants may beused to produce a backcross conversion of inbred pepper line PPL1501that comprises such mutation.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the embodiments areintended to be within the scope of the embodiments.

Gene Editing Using CRISPR

Targeted gene editing can be done using CRISPR/Cas9 technology (Saunders& Joung, Nature Biotechnology, 32, 347-355, 2014). CRISPR is a type ofgenome editing system that stands for Clustered Regularly InterspacedShort Palindromic Repeats. This system and CRISPR-associated (Cas) genesenable organisms, such as select bacteria and archaea, to respond to andeliminate invading genetic material. Ishino, Y., et al. J. Bacteriol.169, 5429-5433 (1987). These repeats were known as early as the 1980s inE. coli, but Barrangou and colleagues demonstrated that S. thermophiluscan acquire resistance against a bacteriophage by integrating a fragmentof a genome of an infectious virus into its CRISPR locus. Barrangou, R.,et al. Science 315, 1709-1712 (2007). Many plants have already beenmodified using the CRISPR system, including Capsicum annuum. See forexample, U.S. Application Publication No. W02014068346 (Gyorgy et al.,Identification of a xanthomonas euvesicatoria resistance gene frompepper (capsicum annuum) and method for generating plants withresistance), Martinelli, F. et al., “Proposal of a Genome Editing Systemfor Genetic Resistance to Tomato Spotted Wilt Virus” American Journal ofApplied Sciences 2014, and Noman, A. et al., “CRISPR-Cas9: Tool forQualitative and Quantitative Plant Genome Editing” Frontiers in PlantScience Vol. 7 November 2016.

Gene editing can also be done using crRNA-guided surveillance systemsfor gene editing. Additional information about crRNA-guided surveillancecomplex systems for gene editing can be found in the followingdocuments, which are incorporated by reference in their entirety: U.S.Application Publication No. 2010/0076057 (Sontheimer et al., Target DNAInterference with crRNA); U.S. Application Publication No. 2014/0179006(Feng, CRISPR-CAS Component Systems, Methods, and Compositions forSequence Manipulation); U.S. Application Publication No. 2014/0294773(Brouns et al., Modified Cascade Ribonucleoproteins and Uses Thereof);Sorek et al., Annu. Rev. Biochem. 82:273-266, 2013; and Wang, S. et al.,Plant Cell Rep (2015) 34: 1473-1476.

Therefore it is another embodiment to use the CRISPR system on inbredpepper line PPL1501 to modify traits and resistances or tolerances topests, herbicides, and viruses.

Introduction of a New Trait or Locus into Inbred pepper line PPL1501

Inbred pepper line PPL1501 represents a new variety into which a newlocus or trait may be introgressed. Direct transformation andbackcrossing represent two important methods that can be used toaccomplish such an introgression. The term backcross conversion andsingle locus conversion are used interchangeably to designate theproduct of a backcrossing program.

Molecular Techniques Using Inbred pepper line PPL1501

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to “alter” (the utilization of up-regulation,down-regulation, or gene silencing) the traits of a plant in a specificmanner. Any DNA sequences, whether from a different species or from thesame species, which are introduced into the genome using transformationor various breeding methods are referred to herein collectively as“transgenes.” In some embodiments, a transgenic variant of inbred pepperline PPL1501 may contain at least one transgene. Over the last fifteento twenty years several methods for producing transgenic plants havebeen developed, and another embodiment also relates to transgenicvariants of the claimed inbred pepper line PPL1501.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthineand others can also be used for antisense, dsRNA and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the embodiments may beproduced by any means, including genomic preparations, cDNApreparations, in-vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

One embodiment is a process for producing inbred pepper line PPL1501further comprising a desired trait, said process comprising introducinga transgene that confers a desired trait to a pepper plant of inbredpepper line PPL1501. Another embodiment is the product produced by thisprocess. In one embodiment, the desired trait may be one or more ofherbicide tolerance, insect tolerance, disease tolerance, or modifiedcarbohydrate metabolism. The specific gene may be any known in the artor listed herein, including: a polynucleotide conferring resistance toimidazolinone, dicamba, sulfonylurea, glyphosate, glufosinate, triazine,PPO-inhibitor herbicides, benzonitrile, cyclohexanedione, phenoxyproprionic acid, and L-phosphinothricin; a polynucleotide encoding aBacillus thuringiensis polypeptide; or a polynucleotide conferringresistance to Tomato spotted wilt virus, Xanthomonas euvesicatoria, Bs2,CARAV1, or CaPMEI1.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Li D. et al, “Establishment of a highly efficienttransformation system for pepper (Capsicum annuum L.)” Plant CellReports, pp 785-788 (2003), and Lee Y.H. et al, “A new selection methodfor pepper transformation: callus-mediated shoot formation” Plant CellReports pp 50-58 (2004). In addition, expression vectors and in vitroculture methods for plant cell or tissue transformation and regenerationof plants are available. See, for example, Gruber, et al., “Vectors forPlant Transformation,” in Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., CRC Press, Inc., Boca Raton, pp.89-119 (1993) and Nakagawa T. et al, “Development of series of gatewaybinary vectors, pGWBs, for realizing efficient construction of fusiongenes for plant transformation” Journal of Bioscience and Bioengineeringpp 34-41 (2007).

A genetic trait which has been engineered into the genome of aparticular pepper plant may then be moved into the genome of anothervariety using traditional breeding techniques that are well known in theplant breeding arts. For example, a backcrossing approach is commonlyused to move a transgene from a transformed inbred pepper line into analready developed inbred pepper line, and the resulting backcrossconversion plant would then comprise the transgene(s).

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to, genes,coding sequences, inducible, constitutive and tissue specific promoters,enhancing sequences, and signal and targeting sequences. For example,see the traits, genes, and transformation methods listed in U.S. Pat.No. 6,118,055.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Amplified Fragment Length Polymorphisms (AFLPs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), Simple SequenceRepeats (SSRs), and Single Nucleotide Polymorphisms (SNPs) may be usedin plant breeding methods utilizing inbred pepper line PPL1501.

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. See Lee J.M et al, “Characterization and moleculargenetic mapping of microsatellite loci in pepper” Theoretical andApplied Genetics pp 619-627 (2004), Kang B.C. et al, “An interspecific(Capsicum annuum xC. Chinese) F2 linkage map in pepper using RFLP andALFP markers” Theoretical and Applied Genetics pp 531-539 (2001), andLefebvre V. et al, “Nuclear RFLP between pepper cultivars (Capsicumannuum L.) Euphytica pp 189-199 (1993).

Randomly Amplified Polymorphic DNAs (RAPDs) and Amplified FragmentLength Polymorphisms (AFLPs) may also be used. See for example Paran, I.et al, “Variation in Capsicum annuum revealed by RAPD and AFLP markers”Euphytica, pp 167-173 (1998), and Llbi, H. “RAPD markers assistedvarietal identification and genetic purity test in pepper, Capsicumannuum” Scientia Horticulturae, pp 211-218 (2003).

SSR technology can be routinely used. See Kwon, Y. et al, “Use of SSRMarkers to Complement Tests of Distinctiveness, Uniformity, andStability (DUS) of Pepper (Capsicum annuum L.) Varieties, Mol. Cells, pp1-8 (2005) and Minamiyama, Y., et al “An SSR-based linkage map ofCapsicum annuum” Molecular Breeding, pp 157-169 (2006).

Single Nucleotide Polymorphisms (SNPs) may also be used to identify theunique genetic composition of the embodiment(s) and progeny varietiesretaining that unique genetic composition. See Jung, J. et al “Discoveryof single nucleotide polymorphism in Capsicum and SNP markers forcultivar identification” Euphytica pp 91-107 (2010), Nicolai, M., et al“Discovery of a large set of SNP and SSR genetic markers byhigh-throughput sequencing of pepper (Capsicum annuum)” Genetics andMolecular Research, pp 2295-2300 (2012), Jeong, H. et al,“Identification of Capsicum species using SNP markers based on highresolution melting analysis” Genome pp 1029-1040 (2010), and Ashrafi,H., et al, “De novo assembly of the pepper transcriptome (Capsicumannuum): a benchmark for in silico discovery of SNPs, SSRs and candidategenes” BMC Genomics (2012).

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome. See Chaim, A.B., et al, “QTL mapping of fruit-related traits inpepper (Capsicum annuum), Theoretical and Applied Genetics, pp 1016-1028(2001) and Rao, G.U., et al, “Mapping of yield-related QTLs in pepper inan interspecific cross of Capsicum annuum and C. frutescens” Theoreticaland Applied Genetics, pp 1457-1466 (2003). QTL markers can also be usedduring the breeding process for the selection of qualitative traits. Forexample, markers closely linked to alleles or markers containingsequences within the actual alleles of interest can be used to selectplants that contain the alleles of interest during a backcrossingbreeding program. The markers can also be used to select for the genomeof the recurrent parent and against the genome of the donor parent.Using this procedure can minimize the amount of genome from the donorparent that remains in the selected plants. It can also be used toreduce the number of crosses back to the recurrent parent needed in abackcrossing program. The use of molecular markers in the selectionprocess is often called genetic marker enhanced selection. Molecularmarkers may also be used to identify and exclude certain sources ofgermplasm as parental varieties or ancestors of a plant by providing ameans of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a pepper plant for which inbred pepper line PPL1501 is a parentcan be used to produce double haploid plants. Double haploids areproduced by the doubling of a set of chromosomes (1N) from aheterozygous plant to produce a completely homozygous individual. Thiscan be advantageous because the process omits the generations of selfingneeded to obtain a homozygous plant from a heterozygous source. Forexample, see, Kele, D. et al, “Effect of Pepper Types on ObtainingSpontaneous Doubled Haploid Plants via Anther culture” HortScience,pp1671-1676 (2015), Olszewska, D. et al, “The assessment of doubledhaploid lines obtained in pepper (Capsicum annuum L.) anther culture”Folia Horticulturae, pp 93-99 (2011), and M. Maluszynski et al. (eds),Doubled Haploid Production in Crop Plants, (2003).

Thus, an embodiment is a process for making a substantially homozygousinbred pepper line PPL1501 progeny plant by producing or obtaining aseed from the cross of inbred pepper line PPL1501 and another pepperplant and applying double haploid methods to the F₁ seed or F₁ plant orto any successive filial generation.

In particular, a process of making seed retaining the molecular markerprofile of inbred pepper line PPL1501 is contemplated, such processcomprising obtaining or producing F₁ seed for which inbred pepper linePPL1501 is a parent, inducing doubled haploids to create progeny withoutthe occurrence of meiotic segregation, obtaining the molecular markerprofile of inbred pepper line PPL1501, and selecting progeny that retainthe molecular marker profile of inbred pepper line PPL1501.

Expression Vectors for Pepper Transformation: Marker Genes

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). Expression vectors includeat least one genetic marker operably linked to a regulatory element (forexample, a promoter) that allows transformed cells containing the markerto be either recovered by negative selection, i.e., inhibiting growth ofcells that do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well-known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or an herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin.

Additional selectable marker genes include Pain1-9a and Pain1-8c whichboth correspond to the group a alleles of the vacuolar acid invertasegene; Pain1prom-d/e; Stp23-8b, StpL-3b, and StpL-3e which originate fromtwo plastid starch phosphorylase genes; AGPsS-9a which is positivelyassociated an increase in plant starch content, starch yield and chipquality, and AGPsS-10a which is associated with a decrease in theaverage plant starch content, starch yield and chip quality; GP171-awhich corresponds to allele 1a of ribulose bisphosphate carboxylaseactivase; and Rca-1a.

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase (Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); Charest, et al., Plant Cell Rep.,8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells, rather than directgenetic selection of transformed cells, for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used marker genes forscreening presumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock, et al., EMBO J., 3:1681 (1984)).

Expression Vectors for Pepper Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions. Many types of promoters are well known in theart.

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment, such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.Many signal sequences are well-known in the art. See, for example,Becker, et al., Plant Mol. Biol., 20:49 (1992); Knox, C., et al., PlantMol. Biol., 9:3-17 (1987); Lerner, et al., Plant Physiol., 91:124-129(1989); Frontes, et al., Plant Cell, 3:483-496 (1991); Matsuoka, et al.,Proc. Natl. Acad. Sci., 88:834 (1991); Gould, et al., J. Cell. Biol.,108:1657 (1989); Creissen, et al., Plant J., b 2:129 (1991); Kalderon,et al., Cell, 39:499-509 (1984); Steifel, et al., Plant Cell, 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes: Transformation

With transgenic plants according to one embodiment, a foreign proteincan be produced in commercial quantities. Thus, techniques for theselection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein can then beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to an embodiment, the transgenic plant provided for commercialproduction of foreign protein is a pepper plant. In another embodiment,the biomass of interest is seed. For the relatively small number oftransgenic plants that show higher levels of expression, a genetic mapcan be generated, primarily via conventional RFLP, PCR, and SSRanalysis, which identifies the approximate chromosomal location of theintegrated DNA molecule. For exemplary methodologies in this regard,see, Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology, CRC Press, Inc., Boca Raton, 269:284 (1993). Mapinformation concerning chromosomal location is useful for proprietaryprotection of a subject transgenic plant.

Likewise, by means of one embodiment, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of pepper, the expression of genes can be altered toenhance disease tolerance, insect tolerance, herbicide tolerance,agronomic, grain quality, and other traits. Transformation can also beused to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to peppers, as well as non-nativeDNA sequences, can be transformed into peppers and used to alter levelsof native or non-native proteins. Various promoters, targetingsequences, enhancing sequences, and other DNA sequences can be insertedinto the genome for the purpose of altering the expression of proteins.The interruption or suppression of the expression of a gene at the levelof transcription or translation (also known as gene silencing or genesuppression) is desirable for several aspects of genetic engineering inplants.

Many techniques for gene silencing are well-known to one of skill in theart, including, but not limited to, knock-outs (such as by insertion ofa transposable element such as Mu (Vicki Chandler, The Maize Handbook,Ch. 118 (Springer-Verlag 1994)) or other genetic elements such as a FRT,Lox, or other site specific integration sites; antisense technology(see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809 (1988) and U.S. Pat.Nos. 5,107,065, 5,453,566, and 5,759,829); co-suppression (e.g., Taylor,Plant Cell, 9:1245 (1997); Jorgensen, Trends Biotech., 8(12):340-344(1990); Flavell, PNAS USA, 91:3490-3496 (1994); Finnegan, et al.,Bio/Technology, 12:883-888 (1994); Neuhuber, et al., Mol. Gen. Genet.,244:230-241 (1994)); RNA interference (Napoli, et al., Plant Cell,2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes Dev., 13:139-141(1999); Zamore, et al., Cell, 101:25-33 (2000); Montgomery, et al., PNASUSA, 95:15502-15507 (1998)), virus-induced gene silencing (Burton, etal., Plant Cell, 12:691-705 (2000); Baulcombe, Curr. Op. Plant Bio.,2:109-113 (1999)); target-RNA-specific ribozymes (Haseloff, et al.,Nature, 334:585-591 (1988)); hairpin structures (Smith, et al., Nature,407:319-320 (2000); U.S. Pat. Nos. 6,423,885, 7,138,565, 6,753,139, and7,713,715); MicroRNA (Aukerman & Sakai, Plant Cell, 15:2730-2741(2003)); ribozymes (Steinecke, et al., EMBO J., 11:1525 (1992);Perriman, et al., Antisense Res. Dev., 3:253 (1993)); oligonucleotidemediated targeted modification (e.g., U.S. Pat. Nos. 6,528,700 and6,911,575); Zn-finger targeted molecules (e.g., U.S. Pat. Nos.7,151,201, 6,453,242, 6,785,613, 7,177,766 and 7,788,044); and othermethods or combinations of the above methods known to those of skill inthe art.

The foregoing methods for transformation may be used for producing atransgenic variety. The transgenic variety could then be crossed withanother (non-transformed or transformed) variety in order to produce anew transgenic variety. Alternatively, a genetic trait that has beenengineered into a particular pepper line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple x by y cross or the process ofbackcrossing depending on the context.

Likewise, by means of one embodiment, agronomic genes can be expressedin transformed plants. More particularly, plants can be geneticallyengineered to express various phenotypes of agronomic interest,including, but not limited to, genes that confer resistance or toleranceto pests or disease, genes that confer resistance or tolerance to anherbicide, genes that confer or contribute to a value-added or desiredtrait, genes that control male sterility, genes that create a site forsite specific DNA integration, and genes that affect abiotic stresstolerance. Many hundreds if not thousands of different genes are knownand could potentially be introduced into a pepper plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a pepper plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125, 1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of one or more embodiments.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of peppers andregeneration of plants therefrom is well-known and widely published.See, Agrawal, S. et al., “Plant regeneration in tissue cultures ofpepper (Capsicum annuum L. cv. Mathania) Plant Cell, Tissue and OrganCulture, Vol. 16(1) pp 47-55 1989; Berljak J. “In vitro plantregeneration from Pepper (Capsicum annuum L. cv. Soroksari') SeedlingExplants” Phyton (Austria) Special issue: “Plant Physiology” Vol. 39(3)pp 289-292 (1999); Ahmad, N., et al., “Improved plant regeneration inCapsicum annuum L. from nodal segments” Biologia Plantarum Vol. 50(4) pp701-704 (2006); and Otroshy, M. et al., “Micropropagation of Pepper(Capsicum annuum L.) Through in vitro Direct Organogenesis” AsianJournal of Biotechnology Vol. 3 pp 38-45 (2010). Thus, another aspect orembodiment is to provide cells which upon growth and differentiationproduce pepper plants having the physiological and morphologicalcharacteristics of inbred pepper line PPL 1501.

Regeneration refers to the development of a plant from tissue culture.The term “tissue culture” indicates a composition comprising isolatedcells of the same or a different type or a collection of such cellsorganized into parts of a plant. Exemplary types of tissue cultures areprotoplasts, calli, plant clumps, and plant cells that can generatetissue culture that are intact in plants or parts of plants, such asembryos, pollen, flowers, seeds, pods, petioles, leaves, stems, roots,root tips, anthers, pistils, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and 5,977,445describe certain techniques, the disclosures of which are incorporatedherein by reference.

Industrial Uses

Pepper has a wide variety of uses in the commodity area. For example,fresh peppers can eaten raw or cooked (fried, baked, boiled, etc).Peppers can also be used to make spices. Thus, a further embodimentprovides for a food product made from a part of the pepper plant varietyPPL1501.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

One embodiment may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

Various embodiments, include components, methods, processes, systemsand/or apparatus substantially as depicted and described herein,including various embodiments, sub-combinations, and subsets thereof.Those of skill in the art will understand how to make and use anembodiment(s) after understanding the present disclosure.

The foregoing discussion of the embodiments has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the embodiments to the form or forms disclosed herein. In theforegoing Detailed Description for example, various features of theembodiments are grouped together in one or more embodiments for thepurpose of streamlining the disclosure. This method of disclosure is notto be interpreted as reflecting an intention that the embodiment(s)requires more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this

Detailed Description

Moreover, though the description of the embodiments has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the embodiments (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the embodiments (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the embodiments unless otherwise claimed.

DESPOSIT INFORMATION

A deposit of the Sakata Seed America, Inc. proprietary inbred pepperline PPL1501 disclosed above and recited in the appended claims ismaintained by Sakata Seed America, Inc., 18095 Serene Drive, MorganHill, Calif. 95037. A deposit will be made with the National Collectionsof Industrial, Food and Marine Bacteria (NCIMB), Ferguson Building,Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UnitedKingdom. Access to this deposit will be available during the pendency ofthis application to persons determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of the seed deposit of the samevariety with NCIMB. The deposit will be maintained in the depository fora period of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedif necessary during that period.

1. A seed of inbred pepper line PPL1501, wherein a representative sampleof seed of said inbred pepper line was deposited under NCIMB No.______.2. A pepper plant, or a part thereof, produced by growing the seed ofclaim
 1. 3. A pepper plant, or a part thereof, having all of thephysiological and morphological characteristics of the plant of claim 2.4. A tissue culture produced from protoplasts or cells from the plant ofclaim 2, wherein said cells or protoplasts are produced from a plantpart selected from the group consisting of leaf, pollen, ovule, embryo,cotyledon, hypocotyl, meristematic cell, callus, root, root tip, pistil,anther, flower, fruit, seed, shoot, stem, and petiole.
 5. A pepper plantregenerated from the tissue culture of claim 4, wherein said plant hasall of the physiological and morphological characteristics of inbredpepper line PPL1501.
 6. A method of producing a hybrid pepper seed,wherein the method comprises crossing the plant of claim 2 with adifferent pepper plant and harvesting the resultant hybrid pepper seed.7. An F₁ hybrid pepper seed produced by the method of claim
 6. 8. An F₁hybrid pepper plant, or a part thereof, produced by growing the seed ofclaim 7, wherein said part is pollen, leaf, ovule, anther, cotyledon,hypocotyl, pistil, root, root tip, flower, fruit, petiole or stem.
 9. Amethod of introducing a desired trait into inbred pepper line PPL1501,wherein the method comprises: (a) crossing a PPL1501 plant, wherein arepresentative sample of seed was deposited under NCIMB No.______, witha plant of another inbred pepper line that comprises a desired trait toproduce progeny plants; (b) selecting one or more progeny plants thathave the desired trait; (c) backcrossing the selected progeny plantswith PPL1501 plants to produce backcross progeny plants; (d) selectingfor backcross progeny plants that have the desired trait andmorphological characteristics of inbred pepper line PPL1501; and (e)repeating steps (c) and (d) two or more times in succession to produceselected third or higher backcross progcny plants that comprise thedesired trait and all of the physiological and morphologicalcharacteristics of pepper PPL1501.
 10. A pepper plant produced by themethod of claim
 9. 11. The pepper plant of claim 10, wherein the desiredtrait is selected from the group consisting of male sterility, herbicidetolerance, insect tolerance, pest tolerance, disease tolerance,environmental stress tolerance, and modified carbohydrate metabolism.12. The pepper plant of claim 11, wherein the desired trait is diseasetolerance and the tolerance is conferred by gene or transgene selectedfrom the group consisting of Bs2, CARA V1, and CaPMEI1.
 13. The pepperplant of claim 11, wherein the desired trait is herbicide tolerance andthe tolerance is conferred to an herbicide selected from the groupconsisting of imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and bcnzonitrile.
 14. The pepper plant ofclaim 11, wherein the desired trait is insect tolerance and the insecttolerance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.
 15. A method of producing a pepper plant comprising a desiredtrait, the method comprising introducing a transgene conferring thetrait into the plant of claim
 3. 16. A pepper plant produced by themethod of claim
 15. 17. A method for producing a seed of a pepper plantderived from inbred pepper line PPL1501, comprising the steps of: (a)crossing the pepper plant of claim 2 with itself or a second pepperplant, a sample of seed of said inbred pepper having been depositedunder NCIMB No.______; and (b) allowing seed of an inbredPPL1501-derived pepper plant to form.
 18. The method of claim 17,further comprising the steps of: (c) selfing a plant grown from saidinbred PPL1501-derived pepper seed to yield additional inbredPPL1501-derived pepper seed; (d) growing said additional inbredPPL1501-derived pepper seed of step (c) to yield additional inbredPPL1501-derived pepper plants; and (e) repeating the crossing andgrowing steps of (c) and (d) to generate at least a first further inbredPPL1501-derived pepper plant.
 19. The method of claim 17, furthercomprising: (f) crossing the further inbred PPL1501-derived pepper plantwith a second pepper plant to produce seed of a hybrid progeny plant.20. A method of producing a pepper fruit, comprising: (a) obtaining theplant according to claim 3, wherein the plant has been cultivated tomaturity; and (b) collecting a pepper from said plant.
 21. A method ofvegetatively propagating a plant of inbred pepper PPL1501, comprisingthe steps of: (a) collecting tissue capable of being propagated from aplant of claim 2; (b) cultivating said tissue to obtain proliferatedshoots; and (c) rooting said proliferated shoots to obtain rootedplantlets.
 22. The method of claim 21, further comprising growing plantsfrom said rooted plantlets.